Notebook

Here you will find our complete labjournal. We documented our daily lab work per week.

June 05-11

Although we obtained the keys of our lab on the first of June we have not yet performed any real experiments. The entire week was spent on setting up the lab, e.g. getting all materials and machines needed. Also we made some media and standard buffers and we have produced our first batch of competent E. coli DH5 α at the end of this week.

June 12-18

General

This week we could finally start doing some real experiments in the lab! We started with optimizing the heat-shock time in the transformation protocol. We did this by transforming our competent cells with DNA from the competent cell test kit with various heat shock times (0-60 seconds). We found that the highest efficiency was obtained when using a heat shock of 30 seconds.

Interlab

Because we did not have fully finished the design of any of the experiments for our own project yet, we started with the Interlab study. As a result we can proudly say that we were the first team to complete the Interlab project this year!! We have successfully transformed all test devices of the interlab studies. Of each of the transformation plates one colony was picked, which was inoculated and grown overnight. After plasmid isolation of the ON cultures restriction analysis with EcoRI showed that all colonies contained the correct plasmids. In the remainder of the week we have completed the measurements according the plate reader protocol. More information about the interlab studies can be found on our interlab page.

June 19-25

General

We have inoculated one of the red colonies from the heatshock-test and isolated the plasmid (pSB1C3:bba_J04450) from it. This plasmid was used to confirm that the restriction enzymes, which were left over from the previous iGEM team of Groningen, worked properly. Moreover this plasmid was also used for cloning parts into the pSB1C3 backbone.

Lactis toolbox

After completing the interlab we started working on the lactis-toolbox. We started with two different parts of this sub-project. The first is the preparation of electro competent L. lactis NZ9000 cells and optimize the transformation protocols for it. The second was the preparation of a pTet-GFP/RFP and a pLac-GFP/RFP construct to test these promotors in L. lactis. From next week progress for this part of the project will be shown under the header ‘promotor testing’. This week we produced our first batch of competent lactis cells and determined a growth curve for NZ9000 in SGM17+glycine medium (see figure below). For the second part we successfully transformed E. coli with pLac (bba_), pTet (bba_), sfGFP (bba_) and a RBS (bba_). Insert figure lactis growth curve

June 26-July 02

dCas9

We have tried to transform our E. coli with the dCas9-omega biobrick (bba_) several times, however all attempts failed until we had no DNA left over. To solve this problem we determined to use the pJWV-102-PL-dCas9 and make the dCas9 biobrick compatible ourselves.

Spacer acquisition

We obtained the gDNA of a clinical isolate of S. pyogenes from …??. Attempts to PCR stukje sebald We can think of two reasons why all PCR reactions failed either this isolate did not have a genomic CRISPR system or the primers bind aspecific to other parts of the DNA. We solved the problem by using plasmids containing the CRISPR operon, which we obtained from the Heler lab [reference].

Lactis toolbox

We have obtained five L. lactis vectors from our supervisor H. Karsens is not in the supervisors list in different host strains (see Table below). The cultures were grown overnight and the plasmids were isolated successfully. table from drive

July 03-09

July 10-16

July 17-23

July 24-30

July 31-August 06

August 07-13

August 14-20

August 21-27

August 28-September 03

September 04-10

September 11-17

September 18-24

September 25-October 01

October 02-08

October 09-15

October 16-22

October 23-29

October 30-November 01

What should this page have?

• Chronological notes of what your team is doing.
• Brief descriptions of daily important events.
• Pictures of your progress.
• Mention who participated in what task.

Inspiration

You can see what others teams have done to organize their notes:

• 2014 ATOMS-Turkiye
• 2014 Tec Monterrey
• 2014 Kyoto
• 2014 Cornell

Next: Future research